Dr. Fernando Flores-Guzman

U.S.A.

Bone marrow dormant tumor cells maintain reactive memory CD8 T cells

Flores-Guzman Fernando1,5, Federico-Guerrero Alfonso2, Morales-Ramirez Claudia A3, Carrasco-Ramirez Liliana4, Umansky Viktor5, Carrasco-Ramirez Elba6

1. Surgery Department, University of Colorado, Denver Colorado, USA
2. Microbiology Department, School of Medicine, UNAM, Mexico City
3. General Hospital Dr. Enrique Cabrera, Mexico City
4. School of Political and Social Sciences, UNAM, Mexico City
5. German Cancer Research Center (DKFZ), Heidelberg, Germany
6. Microscopy Department, School of Medicine, UNAM, Mexico City

Abstract

Background

The hypothesis of the cancer stem cell (CSC) suggests that neoplastic clones are maintained by a fraction of tumor cells and represent disseminated dormant tumor cells. We used a ret transgenic mouse spontaneous melanoma model, in which 25% of transgenic mice develop skin tumors with metastases in lymph nodes (mLN), liver, lungs and bone marrow (BM). Mice older than 20 weeks without macroscopic tumors contain in the BM tyrosinase related protein (TRP)-2-specific effector memory CD8+T cells and show no further melanoma progression. This suggests a potential role of dormant tumor cells in the maintenance of memory CD8+T cells.

Methods

We used a C57BL/6 mice expressing the human RET oncogene in melanocytes. As a control, we used C57BL/6 wild type mice that show no expression of the human RET oncogene. Single tumor cell suspensions were fixed and permeabilized using Foxp3 fixation/permeabilization kit. Then, cells were incubated with antibodies for flow cytometry. Acquisition was performed by six-color flow cytometry using FACSCanto. The Immunofluorescence for CD133 and TRP-2 in primary melanoma lesions were stained with primary anti-CD133 antibodies labeled with Alexa-Fluor 555/594. For the staining of TRP-2, the samples were incubated with rabbit anti-mouse antibodies against TRP-2, then with second secondary antibodies labeled with Alexa-Flour-488. The acquisition was performed using a confocal laser scanning microscope LSM-710, and data were analyzed by ZEN software. BM specimens were isolated from femurs by aspiration, and 1×105 BM cells/sample were prepared using cytospin technique.

Results

TRP-2+CD133+ melanoma cells represent less than 1.5% of all cells in primary skin tumors and mLN. Most of these cells were Ki67-negative, thereby these cells exist in a dormant state. The dormant state of TRP-2+CD133+ melanoma cells was confirmed on the negative expression of Ki67 and PCNA. We found that TRP-2+Ki67-negative melanoma cells were co-localized with memory CD8+T cells both in mice without and with macroscopic tumors and the rate of memory CD8+ T cells interacting with TRP-2 +Ki67-negative melanoma cells was less than 15% in the BM. Certain IFN-gamma-producing CD8+T cells interacted either with single TRP-2+ melanoma cells or the smallest cluster of melanoma cells (2-5 TRP-2+ cells), which two TRP-2-specific CD8+T cells produced perforin, but none of them were co-localized either with TRP-2+ melanoma cells or TRP-2+CD133+ melanoma cells.

Conclusions

Our data demonstrates the existence of a subpopulation of CD133+ melanoma cells in ret transgenic mice and dormant TRP-2+ melanoma cells are able to interact with CD8+T cells in the BM of tumor-bearing mice.